SHIP-deficient CD4⁺CD25⁺ T_regs exhibit normal immunoregulatory capacity in vivo. C57BL/6J SCID hosts received 4 × 10⁵ CD3⁺CD4⁺CD25⁻ T cells from WT C57BL/6J donors by intraperitoneal injection on day 1 (n = 7). Where indicated, the WT effector CD3⁺CD4⁺CD25⁻ T cells were coinjected with 7 × 10⁴ CD3⁺CD4⁺CD25⁺ T_regs from WT or SHIP^−/− C57BL/6J donors into C57BL/6J SCID hosts (n = 9). In parallel, a control group of C57BL/6J SCID hosts received a PBS injection (n = 5). Disease and weight were monitored every other day. (A) Analysis of the rate of weight change over the course of the study (3 months) in the cohorts that received WT effector CD3⁺CD4⁺CD25⁻ T cells only (labeled WT CD25⁻), or effector WT CD3⁺CD4⁺CD25⁻ T cells with WT or SHIP^−/−CD3⁺CD4⁺CD25⁺ T_regs (labeled WT CD25⁻ and WT CD25⁺ or WT CD25⁻ and −/− CD25⁺, respectively), or PBS. The weight change depicted was determined by converting each actual weight to a percentage of that mouse's initial weight. Each line depicts the average weight change for the specified cohort (P < .001, WT CD25⁻ vs WT CD25⁻ and WT CD25⁺, WT CD25⁻ vs WT CD25⁻ and SHIP^−/− CD25⁺, and for WT CD25⁻ vs PBS; P > .1 for WT CD25⁻ and WT CD25⁺ vs WT CD25⁻ and SHIP^−/− CD25⁺, WT CD25⁻ and WT CD25⁺ vs PBS, and for WT CD25⁻ and SHIP^−/− CD25⁺ vs PBS). (B) Histologic appearance of the colon (hematoxylin and eosin, ×200) from a mouse in the WT CD25⁻ (top left; HPS = 3), WT CD25⁻ and WT CD25⁺ (top right; HPS = 1), WT CD25⁻ and −/− CD25⁺ (bottom right; HPS = 1), and PBS (bottom left; HPS = 0) cohorts. Histology micrographs were taken using a Leica DMLB microscrope (N PLAN 20×/0.40, total magnification ×200, at room temperature), and a SPOT Insight QE Model 42 camera with Spot Advanced acquisition software (Diagnostic Instruments, Sterling Heights, MI). (C) Box and whisker plots and table summarizing the histopathology scores (HPS) given to the hosts within each cohort. The HPS was determined by grading the histologic appearance of the colon using the following criteria: grade 0 indicates unaffected proximal colon; grade 1, mild leukocyte infiltration of the lamina propria (not shown); grade 2, moderate leukocyte infiltration of the lamina propria, mild reduction of goblet cells, and mild crypt epithelial regenerative hyperplasia; grade 3, marked leukocyte infiltration beyond the muscularis mucosa into a thickened submucosa, goblet cell depletion, and epithelial regenerative hyperplasia with atypia; grade 4, marked transmural leukocyte infiltration deep into a thickened submucosa and tunica muscularis with increased vascular density, marked goblet cell loss, and epithelial regenerative hyperplasia with atypia used to determine histopathologic score (HPS). P < .001, WT CD25⁻ versus WT CD25⁻ and WT CD25⁺, WT CD25⁻ versus WT CD25⁻ and SHIP^−/− CD25⁺, and for WT CD25⁻ versus PBS; P > .5 for WT CD25⁻ and WT CD25⁺ versus WT CD25⁻ and SHIP^−/− CD25⁺.